In addition, in order to investigate the effect of particle size on the dissolution and oral bioavailability of the trans-resveratrol, two microparticles with different sizes (1.94 μm and 18.75 μm) were prepared using two different milling processes, and compared to trans-resveratrol nanoparticles prepared by the SAS process. The solid-state properties of pure trans-resveratrol particles were characterized. By increasing the percentage of dichloromethane in the solvent mixtures, the mean particle size of trans-resveratrol was decreased, whereas its specific surface area was increased. The particle size could thus be controlled by solvent composition. Trans-resveratrol nanoparticle with a mean particle size of 0.17 μm was prepared by the SAS process using the ethanol/dichloromethane mixture at a ratio of 25/75 (w/w). The in vitro dissolution rate of trans-resveratrol in fasted state-simulated gastric fluid was significantly improved by the reduction of particle size, resulting in enhanced oral bioavailability in rats. The absolute bioavailability of trans-resveratrol nanoparticles was 25.2%. The maximum plasma concentration values were well correlated with the in vitro dissolution rate. These findings clearly indicate that the oral bioavailability of trans-resveratrol can be enhanced by preparing pure trans-resveratrol nanoparticles without additives  by the SAS process. These pure trans-resveratrol nanoparticles can be applied as an active ingredient for the development of health supplements, pharmaceutical products, and cosmetic products.The aim of this study was to prepare pure  Trans-resveratrol, a phytochemical with strong antioxidant property, is naturally found in grapes, berries, and peanuts. The benefits of trans-resveratrol have been reported in various diseases, including cancer, cardiovascular diseases, diabetes mellitus, neurological diseases, obesity, and other conditions associated with oxidative stress and inflammation [trans-resveratrol is converted into cis-resveratrol by light and has a very low aqueous solubility [max) values obtained after oral administration at a dose of 40-mg trans-resveratrol were approximately 10-fold and 8.5-fold higher in the capsule containing solubilized resveratrol in polysorbate 20 and polyglyceryl-3-dioleate mixture than in powder capsule , respectively [trans-resveratrol produced higher AUC (5.0-fold increase) and Cmax (10.6-fold increase) than the equivalent dose of the original powder [trans-resveratrol, the oral bioavailability of the micronized powder (particle size less than 5 μm) was significantly higher than that of the nonmicronized powder, with approximately 3.6-fold increases in the Cmax values at 14 days [trans-resveratrol must be administered for long periods. Minimizing the use of additives such as surfactants (polysorbates and docusate sodium) can reduce its side effects.ammation ,2,3,4,5.lubility ,7. Furthlubility ,9. To dalubility ,17,18,19lubility ,21,22. Ptrans-resveratrol nanoparticles without the additives  by using the supercritical antisolvent (SAS) process and alcohol and dichloromethane mixtures. Herein, the solid-state properties of the pure trans-resveratrol nanoparticles were characterized. Further, particle size reduction of trans-resveratrol was performed using a Fitz mill and an air jet-mill, and a dissolution and pharmacokinetic study was conducted to investigate the effect of particle size on the dissolution and oral bioavailability of trans-resveratrol.Particle size reduction for poorly water-soluble phytochemicals is an important process for increasing the dissolution rate and, consequently, the oral bioavailability of compounds ,24,25,26Trans-resveratrol was purchased from Ningbo Liwah Pharmaceutical Co., Ltd. . United States Pharmacopeia (USP) reference standard of trans-resveratrol, dimethyl sulfoxide (DMSO), and polyethylene glycol (PEG) 300 were purchased from Sigma-Aldrich Co. . Methanol, ethanol, and dichloromethane of high-performance liquid chromatography (HPLC) grade were purchased from Honeywell Burdick and Jackson . Carbon dioxide (99.99% purity) was supplied from Hana Gas Co., Ltd. .trans-resveratrol in the solvents, the solubility of trans-resveratrol in alcohol (methanol or ethanol) and dichloromethane mixtures was determined by the shake-flask method through solid–liquid equilibrium [w/w). Excess trans-resveratrol  was mixed with the solvents in amber vials via vortex for 15 min and sonication in an ultrasonic bath  for 45 min. The amber vials were shaken at 60 rpm in a water-bath mechanical shaker  at 25 °C for at least 24 h. For HPLC analysis, the sample was filtered using a 0.2 μm syringe filter to obtain a clear supernatant from a saturated solution. Thereafter, the filtrate was placed in a preweighed volumetric flask. The concentration of trans-resveratrol was determined using a Prominence HPLC-ultraviolet (HPLC-UV) system  after dilution with methanol. Trans-resveratrol was separated by Gemini C18 column . The mobile phase consisted of acetonitrile/water . The flow rate was 0.8 mL/min, column temperature was 30 °C, and the detection wavelength was 303 nm. All solubility measurements were repeated three times.To understand the solubility behavior of ilibrium ,33,34. Btrans-resveratrol nanoparticles were prepared by the SAS process, as previously reported [trans-resveratrol (30 mg/g) was dissolved in alcohol (methanol or ethanol) and dichloromethane at different weight ratios  in an amber vial. Carbon dioxide liquefied with a cooler was heated to a desired point (40 °C) using a heat exchanger, and then injected into the particle formation vessel (1 L) through a two-way nozzle using a syringe pump . Once a pressure of 150 bar and temperature of 40 °C were achieved and equilibrium was reached, supercritical carbon dioxide was delivered at a rate of 40 g/min and the sample solution was simultaneously injected at a rate of 0.5 g/min using an HPLC pump . Upon injecting the sample solution, only carbon dioxide was further pumped at the same rate to remove the residual solvent that could dissolve the prepared particles. The SAS system was slowly depressurized to atmospheric pressure. The generated particles were collected on the walls and bottom of the particle formation vessel.Pure reported ,36,37. Ttrans-resveratrol. To generate trans-resveratrol microparticles with the mean particle size of approximately 20 μm, raw trans-resveratrol with a mean particle size of 108.94 μm was milled in the Fitz mill  under the following conditions: speed, 500–1000 rpm; feed rate, 0.5 g/min. To obtain trans-resveratrol microparticles with a mean particle size of approximately 2 μm, continuous grinding using an air jet-mill  was conducted under the following process conditions: injection pressure, 8 bar; grinding pressure, 8 bar; and feed rate, 0.5 g/min.Two different milling processes using a Fitz mill and an air jet-mill were applied for particle size reduction of raw trans-resveratrol after the SAS process and milling process was validated by HPLC analysis. The sample solution and standard solution were prepared by dissolving the processed trans-resveratrol and USP reference standard in methanol at 100 μg/mL concentration, respectively. A 10-μL volume of the solution was injected into a Prominence HPLC-ultraviolet (HPLC-UV) system  as described above. Residual solvents of trans-resveratrol after the SAS process were measured by a gas chromatograph (GC)  coupled with head space sampler  and flame ionization detector. Analysis was performed as previously described with slight modifications [The purity of ications . The fustrans-resveratrol nanoparticles and microparticles was observed using a scanning electron microscope . Electrically conductive samples were prepared by gold coating in vacuum for 1 min, before observation. Samples were visualized at an accelerating voltage of 5 kV.The morphology of the trans-resveratrol nanoparticles and microparticles were determined using laser diffraction  according to the Mie scattering theory. Samples were sufficiently dispersed in water, and all particle size measurements were repeated three times.For the nanoparticles, the z-average size and polydispersity index (PI) were measured using ELSZ-1000  according to the dynamic light scattering (DLS) method. In addition, particle size and size distribution of trans-resveratrol nanoparticles and microparticles was determined using the Micromeritics TriStar II 3020 instrument  based on the Brunauer–Emmett–Teller method with the nitrogen adsorption analysis after degassing samples with helium (purity > 99.99%).The specific surface area of trans-resveratrol samples ranging from 3 to 4 mg were accurately weighed and sealed in an aluminum pan. The same hermetic aluminum pan without sample was used as a reference. The measurements were carried out under nitrogen purge (300 mL/min) in the temperature range of 0 to 300 °C, with a heating rate of 10 °C/min. All measurements for each sample were performed in triplicate.DSC measurements were conducted with a Discovery DSC 25 instrument connected to an RSC90 cooling system . High purity indium and aluminum oxide sapphire were used to calibrate temperature and heat capacity. The PXRD analysis was carried out using an X-ray diffractometer , with Cu-Kα radiation at a voltage of 40 kV and a current of 40 mA. The samples were scanned from 5° to 50° (2θ), with a scanning speed of 3°/min and a step size of 0.01°.trans-resveratrol. To investigate the effect of particle size on dissolution and to derive the relationship between in vitro dissolution rate and in vivo pharmacokinetic parameters, we conducted dissolution testing of trans-resveratrol nanoparticles and microparticles in fasted state-simulated gastric fluid (FaSSGF) and fasted state-simulated intestinal fluid (FaSSIF) as biorelevant dissolution media. First, the solubility of trans-resveratrol  in FaSSGF and FaSSIF was determined at 37 °C as described in the solubility measurement method above. FaSSGF and FaSSIF were prepared with sodium chloride, hydrochloric acid, and Biorelevant powder  and sodium hydroxide, sodium phosphate monobasic, sodium chloride, and Biorelevant powder, respectively, according to the method retrieved from Biorelevant.com. The dissolution test was carried out in accordance with the USP paddle method using a Varian VK 7010 dissolution system  at 50 rpm and 37 ± 0.5 °C. To neglect the effect of wettability, trans-resveratrol nanoparticles and microparticles were dispersed in water at 50 mg/mL prior to the dissolution study. A sample of 50 mg trans-resveratrol was placed in 900 mL of dissolution media (FaSSGF). Dissolved trans-resveratrol was quantified using Pion Rainbow spectrometers with 2-mm path length UV fiber optic probes  at a wavelength of 306 nm. Data were collected every 1 min for 60 min, and all dissolution measurements were repeated six times.Dissolution under the physiological conditions of the gastrointestinal tract is the first process in the oral absorption of trans-resveratrol nanoparticles and microparticles were obtained following their oral administration to male Sprague–Dawley (SD) rats. The animal study protocol complied with the institutional guidelines for the care and use of laboratory animals and was approved by the ethics committee of Kyungsung University (No. 19-008A). Twenty-four male SD rats  were divided into four treatment groups (n = 6 per group). Three groups received either trans-resveratrol nanoparticles (0.17 μm) prepared by the SAS process, trans-resveratrol microparticles (1.94 μm) milled by the air jet-mill, and trans-resveratrol microparticles (18.25 μm) milled by the Fitz mill at a dose of 50 mg/kg using an oral dosing apparatus. Each particle was dispersed in 1 mL of water immediately before dosing [trans-resveratrol nanoparticles and microparticles, one group was intravenously administered via the tail vein a dose of 15 mg/2.5 mL/kg  [trans-resveratrol in plasma was determined using a previously reported method and under HPLC analysis conditions [0→8h was calculated using the linear trapezoidal method. Cmax and the time required to reach Cmax (Tmax) were directly obtained from plasma data.To investigate the effect of particle size on bioavailability, the pharmacokinetic parameters of e dosing . To esti:85 v/v) . Serial nditions ,41. The n = 3, 4, or 6). One-way analysis of variance followed by the least significant difference test and the Student–Newman–Keuls test was performed using SPSS .Data are expressed as mean ± standard deviation  and dichloromethane mixtures at different weight ratios  and the measured data were graphically plotted . As shown in trans-resveratrol.To prepare the  plotted . The soltrans-resveratrol nanoparticles using the SAS process and 30 mg/g of trans-resveratrol to investigate the effect of solvent composition  on particle size and morphology. As shown in trans-resveratrol precipitated from the methanol–dichloromethane mixtures had smaller nanoparticles than those generated from methanol. By increasing the percentage of dichloromethane in the solvents, the mean particle size decreased and the specific surface area increased. The PI values for trans-resveratrol precipitated from the methanol/dichloromethane mixtures at ratios 25:75 and 50:50 (w/w) were below 0.2, indicating narrow particle size distribution [trans-resveratrol was reduced. In particular, the trans-resveratrol nanoparticles prepared at a ratio of 25:75 (ethanol:dichloromethane) had a mean particle size of 151.2 nm, with 60.23 m2/g. Additionally, it had the smallest particle size among the prepared samples.Based on the solubility data, we prepared ribution ,43. Coheribution ,45 reportrans-resveratrol in all processed samples were the same as those of the unprocessed raw trans-resveratrol (p > 0.05), which means that there was no difference in crystal polymorphism and crystallinity between nanoparticle samples prepared by the SAS process. The process yield was above 85% for all experiments. All trans-resveratrol nanoparticles prepared using the SAS process exhibited low residual solvents; methanol < 20 ppm, ethanol < 20 ppm, and dichloromethane < 10 ppm according to the residual solvent analysis. The purity (%) of all trans-resveratrol nanoparticles was above 99.3%, indicating no degradation and conversion to cis-resveratrol during the SAS process. Therefore, pure trans-resveratrol nanoparticles were successfully prepared using the SAS process with alcohol and dichloromethane and their particle size could be controlled by solvent composition. Based on the above results, trans-resveratrol nanoparticles prepared at a ratio of 25:75  were selected as the best sample for further investigation.As shown in trans-resveratrol microparticles were prepared using a Fitz mill and an air jet-mill in contrast to the nanoparticles prepared by the SAS process. As a result, the particle size of raw trans-resveratrol with a mean particle size of 108.94 μm was decreased to 18.25 μm by the milling process using the Fitz mill and 1.94 μm by the air jet-mill. In contrast, the trans-resveratrol nanoparticles with mean particle sizes of 0.17 μm and 60.23 m2/g were prepared by the SAS process using the ethanol: dichloromethane mixture at a ratio of 25:75. The physicochemical properties of the trans-resveratrol nanoparticles and microparticles are summarized in trans-resveratrol nanoparticles and microparticles were not significantly different (p > 0.05) to those of the unprocessed raw trans-resveratrol  was superior to that in FaSSGF (pH 1.5) and could be attributed to micellar solubilization instead of pH as a result of the higher concentration of sodium taurocholate and lecithin; this is because trans-resveratrol exhibited a similar solubility in the pH range of 1.2–6.8 [trans-resveratrol was applied for solubilization in 900 mL of FaSSGF or FaSSIF. The dissolution profiles of the trans-resveratrol nanoparticles and microparticles in FaSSGF and FaSSIF are shown in trans-resveratrol was affected by its particle size in both FaSSGF and FaSSIF. As a nanoparticle (0.17 μm), trans-resveratrol was approximately 90% dissolved within 3 min; however, as a microparticle (18.25 μm), only ~50% dissolved at 60 min in both FaSSGF and FaSSIF. This enhancement in the dissolution rate is a consequence of the increase in surface area and decrease in diffusion layer thickness by particle size reduction. For further quantitative analysis, the Hixson–Crowell cube root equation was applied to estimate the resveratrol release rate and simulated 50% dissolution time (t50%) [t50%) for the trans-resveratrol nanoparticles and microparticles were increased and decreased, respectively, as the particle size was reduced.To investigate the effect of particle size on dissolution and derive the relationship between in vitro dissolution rate and in vivo pharmacokinetic parameters, we carried out a dissolution test with the  1.2–6.8 ,46. To pe (t50%) . As showtrans-resveratrol nanoparticles and microparticles were obtained following their oral administration to male SD rats  employed for oral administration in rats [To investigate the effect of particle size on the bioavailability, the pharmacokinetic parameters of  SD rats . In addiveratrol . Nonethe in rats ,49,50,51trans-resveratrol. Thus, the dissolution data obtained in FaSSGF were employed to establish the relationship between in vitro dissolution rate and in vivo pharmacokinetic parameters  and dichloromethane. We demonstrated that their particle size is controlled by solvent composition. Further, we revealed that the in vitro dissolution rate of trans-resveratrol is significantly improved by a reduction in particle size, thereby enhancing the oral bioavailability. These findings clearly indicate that the oral bioavailability of trans-resveratrol can be enhanced by preparing pure trans-resveratrol nanoparticles without additives  by the SAS process. This pure trans-resveratrol nanoparticle can be used as an active ingredient in the development of health supplements, pharmaceutical products, and cosmetic products.Pure


Astroviridae family. Infection of classic HAstVs is one of the most common causes of acute viral gastroenteritis . There is a lack of data on the prevalence and genetic characterization of classic HAstVs in acute viral gastroenteritis in the whole population. This study aimed to investigate the epidemiological trend, genotypes, viral co-infections, and viral loads of classic HAstVs in Shanghai, China, from January 2015 to December 2016.Human astroviruses (HAstVs) are single-stranded RNA viruses of the A total of 6,051 non-redundant stool samples were collected in outpatients with acute diarrhea in Shanghai from January 2015 to December 2016. One-step real-time RT-PCR was used for screening viral diarrhea, including rotavirus A, rotavirus B, rotavirus C, norovirus genotype I and II, classic human astroviruses, and sapovirus. Real-time PCR was used for screening human enteric adenoviruses. Conventional RT-PCR was used for the amplification of viral fragments for genotyping. PCR products were sequenced and used for the construction of phylogenetic trees.The detection rate of classic HAstVs was 1.55% . The prevalence of HAstV infection displayed a typical winter/spring (December to March) seasonality and was highest in the 5–14 age group. Eighty-six samples were genotyped, which revealed HAstV-1 as the most prevalent genotype, followed by HAstV-5, HAstV-4, HAstV- 2, HAstV-8, and HAstV-3. There was a dramatic rise in the prevalence of HAstV-4 from December 2015 to March 2016, and the viral loads of HAstV-4 were significantly higher than those of other genotypes. Among the mixed infection samples, noroviruses were found to be the most frequently co-infected enteric viruses with HAstV.Multiple genotypes of classic HAstVs circulated in Shanghai from January 2015 to December 2016. For the first time, HAstV-3\4\5\8 were detected in Shanghai. Astroviridae family. Classic HAstVs are among the most important viruses causing acute viral gastroenteritis worldwide (Human astroviruses (HAstVs) are small, non-enveloped, positive-sense single-stranded RNA viruses of the orldwide . Infectiorldwide . Mixed iorldwide . In suscorldwide .The genome of HAstVs harbors three open reading frames (ORFs), namely, ORF1a, ORF1b, and ORF2. ORF1a and ORF1b encode non-structural proteins (nsPs) involved in RNA transcription and replication, while ORF2 encodes the capsid proteins. According to the International Committee on Taxonomy of Viruses (ICTV), HAstVs are classified into three divergent groups, namely, classic HAstVs , HAstV-MLB (MAstV 6), and HAstV-VA/HMO (MAstV 8 and MAstV 9) .Classic HAstVs segregate into eight genotypes (HAstV-1–HAstV-8), and each corresponds to an individual serotype. Lineages or subtypes have been identified in HAstV-1–HAstV-6, and there are a total of 20 lineages  . A new lMost studies of viral diarrhea etiology focus on children aged under 5 years, which is a partial representation of the general population . FurtherIn 2012, Shanghai Municipal Centers for Disease Control and Prevention (CDC) established the Shanghai diarrhea comprehensive surveillance system (SDCSS) and has been taking on continuous surveillance since then. In this study, we analyzed the available SDCSS data from January 2015 to December 2016 to investigate the epidemiological trend, genotypes, viral co-infections, and viral loads of HAstV-associated gastroenteritis in all age groups in Shanghai. The study revealed that multiple genotypes of classic HAstVs circulated in Shanghai and HAstV-3\4\5\8 were discovered in Shanghai for the first time.Diarrheal patients were defined as those who visited the enteric disease clinics of sentinel hospitals, with three or more loose or liquid stools per day. HAstV-affected patients were defined as those whose stool samples were positive for HAstV PCR detection at the initial screening, including patients with viral co-infections.A total of 6,051 non-redundant fecal samples were collected using the probability proportional to size sampling method across 22 sentinel hospitals of SDCSS in outpatients with acute diarrhea in Shanghai from 2015 to 2016. The SDCSS system consists of Shanghai municipal CDC, 16 district CDCs, and 22 sentinel hospitals. Sample information was collected through an online questionnaire, including age, gender, treatment time (time of onset), season, and clinical symptoms. The study protocol was reviewed and approved by the Human Research Ethics Committee of the Shanghai Municipal Centers for Disease Control and Prevention.Viral nucleic acids  were extracted from 200 μL of 10% fecal suspension in phosphate-buffered saline. The extraction was done using a MagNA Pure LC Total Nucleic Acid Isolation Kit according to the manufacturer’s instructions . Viral nucleic acids were eluted in 50 μL of elution buffer and stored at −80°C immediately.Classic HAstVs, norovirus genogroup I and II, sapovirus, and rotavirus A, rotavirus B, rotavirus C were detected using real-time reverse transcription PCR (RT-PCR) assays. Enteric adenoviruses were detected using real-time PCR. All assays were performed using the respective commercial kits according to the manufacturer’s instructions . Primer information is listed in TM III One-Step RT-PCR System with a PlatinumTM Taq DNA Polymerase Kit . Briefly, 10 μL of extracted viral RNA was added to 40 μL of a reaction mixture containing 25 μL of 2 × Reaction Mix, 1 μL sense primer (10 μM), 1 μL anti-sense primer (10 μM), 1 μL SuperScriptTM III RT/PlatinumTM Taq Mix, and 12 μL autoclaved distilled water. The mixture was reverse transcribed for 30 min at 55°C and 2 min at 94°C. PCR amplification was performed under the following thermal cycling conditions: initial denaturation at 94°C for 2 min, followed by 40 cycles of amplification with denaturation at 94°C for 15 s, primer annealing at 55°C for 30 s, and an extension reaction at 68°C for 1 min and then a final extension at 68°C for 5 min.Conventional RT-PCR was used for confirmation of classic HAstVs and subtyping. RT-PCR was performed using a SuperScriptViral loads of HAsVs in fecal samples were measured using quantitative real-time RT-PCR with primers and probe targeting ORF1b . Briefly, 5 μL of sample RNA was mixed with 20 μL of a reaction buffer containing reaction master mix, sense and anti-sense primers, probe, and nuclease-free water. Real-time RT-PCR was performed under the following thermal cycling conditions on a LightCycler 480 real-time PCR instrument II : initial reverse transcription at 45°C for 15 min for the synthesis of cDNA, followed by 45 cycles of amplification with denaturation at 94°C for 15 s, and annealing and extension at 62°C for 60 s. Fluorescence readings were collected and analyzed with LightCycler 480 software . The genome copy numbers of HAstVs were determined based on a standard curve prepared with 10-fold serial dilutions of plasmid DNA provided in the commercial kit .1.The PCR products obtained from viral  positive specimens were subjected to Sanger sequencing . The sequences were edited using BioEdit software and compared with reference sequences in the NCBI GenBank database using the BLAST serverThe reference strains from GenBank representing different HAstV genotypes were included in the phylogenetic analysis. All the selected sequences  were ali2 test and a Fisher exact with a two-tailed method. Differences between HAstV genotypes and the viral loads in samples were determined using analysis of variance (ANOVA). P-values of <0.05 were considered statistically significant.All calculations were conducted using Microsoft Excel 2013 and SPSS software v19.0 . We determined whether there were statistically significant differences between the different age groups with detected virus using a χA total of 6,051 non-redundant stool samples from diarrheal patients were collected from January 2015 to December 2016. Viral screening using one-step real-time RT-PCR indicated that 28%  of the samples contained at least one virus, and 124 samples had two or three viruses. Norovirus genogroup II was the most frequently detected (15.17%), followed by rotavirus A (7.12%), norovirus genogroup I (2.64%), sapovirus (2.53%), HAstVs (1.55%), enteric adenovirus (1.04%), and rotavirus C (0.17%) . No rotap = 0.005) (n = 20) and landed at the lowest level in July (n = 0)   (Supplem= 0.005) . A typic (n = 0) .2 to 7.53 × 1011 per gram of stool sample (p < 0.001). The results indicate that multiple genotypes were circulating in Shanghai from January 2015 to December 2016.HAstV genomic fragments  were successfully amplified from 90 of the 94 HAstV-positive samples initially screened. Phylogenetic analysis of the sequences of these fragments revealed six HAstVs genotypes (HAstV-1–5 and HAstV-8) among 86 samples and four non-subtyped samples . HAstV-6l sample . The meaThe highest percentage of mixed infection by tested enteric viruses were observed in samples positive for norovirus genogroup I  and sapovirus  . For HAsAmong the co-infected cases, HAstV-1 appeared as the most prevalent (7/26), followed by HAstV-5 and HAstV-4 (both were 6/26), HAstV-2, and HAstV-8 (both were 2/26), HAstV-3 (1/26), and two non-genotyped , which iAcute viral gastroenteritis is one of the most common infectious diseases worldwide. Human astrovirus is currently recognized as a major cause of sporadic gastroenteritis in both children and adults. In China, most published studies have mainly focused on HAstV infections of children, especially children under 5 years old. This study was the first report on the epidemiological trends, genotypes, viral co-infections, and viral loads of HAstV infections of the whole population in Shanghai, based on citywide diarrheal surveillance data from January 2015 to December 2016.Out of 6,051 stool samples, the HAstV positive rate was 1.55% . The detection rates of HAstV varied in previous studies on the data from China, ranging from 1.8 to 13.6%, depending on the age group and detecting methods . InteresOur study showed a typical cold season distribution of HAstV infection with a single peak. The peak from December 2015 to April 2016 was much higher than the previous one. Two peaks of HAstV detection rate were observed in Germany, between February and May and between September and December . In KoreSix genotypes were found to circulate during 2015–2016, with HAstV-1 being predominant, followed by HAstV-5, HAstV-4, HAstV-2, HAstV-8, and HAstV-3. Except for HAstV-1a and 1b, all other HAstVs are of a single subtype/lineage. HAstV-5\4\2\8\3 have not been reported in Shanghai. The detection of these genotypes in this study indicates that multiple HAstVs were circulating in Shanghai. It would be interesting to monitor the frequency of these genotypes in the Shanghai population in the future. Like other recent studies , HAstV-6Notably, the peak of HAstV-induced cases in 2016 was almost five times higher than in the preceding year. A rapid rise in HAstV-4 cases was observed during the high HAstV peak season from December 2015 to April 2016. Furthermore, HAstV-4-positive samples showed a high viral load. Multiple factors might have contributed to this result. Compared with the 2015 data, both the case number and detected genotypes of HAstV infections had increased over the same period last year, and HAstV-4 in particular had the most obvious change. The HAstV-4 strains observed in this study may be a variant because they exhibited a characteristically high stool shedding. In addition to clarifying the relationship between this variant and the severity of the disease, it is also necessary to study its infectiousness. As is well known, norovirus infections are a leading cause of acute gastroenteritis worldwide in persons of all age groups. Interestingly, the cases of norovirus infections in January 2016 were almost five times lower than in January 2015 during the same period based on SDCSS data . In contThis study has several limitations. First, non-classic HAstVs  were not detected. The primers for the initial screening of sentinel hospitals were not suitable for non-classic HAstV detection. Future screening should include the detection of these non-classic HAstVs. Second, no diarrhea-free control group was included in the study. Results from the Hebei Province of China among children showed a high HAstV detection in asymptomatic control children , suggestIn conclusion, this is the first report on the epidemiological characteristics of classic human astroviruses in Shanghai from January 2015 to December 2016. Our study pointed out that multiple classic HAstV genotypes circulated simultaneously in Shanghai, and that for the first time, HAstV-3\4\5\8 were detected in Shanghai. There was a sharp increase in HAstV-4 cases during the winter of 2015 and the spring of 2016.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by the Shanghai Municipal Center for Disease Control and Prevention Ethical Review Committee (No. 2017-26). Affiliation: Shanghai Municipal Center for Disease Control and Prevention. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.YX and XZ designed this study. LW, QL, JL, HW, and Z-aY analyzed the data. XK and XQC collected the samples. LW, WW, and XXC contributed the samples testing. LW, ZT, QL, XZ, and YX wrote this manuscript. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Klebsiella species serotypes to aid in outbreak surveillance. A reliability score (estimated based on the accuracy of a specific K-type prediction against the dataset of 141 distinct K-types) average (ARS) that reflects the specificity between the Klebsiella species capsular polysaccharide biosynthesis and surface expression proteins, and their K-types has been established. ARS indicates the following order of potency in accurate serotyping: Wzx (ARS = 98.5%),Wzy (ARS = 97.5%),WbaP (ARS = 97.2%),Wzc (ARS = 96.4%),Wzb (ARS = 94.3%),WcaJ (ARS = 93.8%),Wza (ARS = 79.9%) and Wzi (ARS = 37.1%). Thus, Wzx, Wzy and WbaP can give more reliable K-typing compared with other proteins. A fragment-based approach has further increased the Wzi ARS from 37.1% to 80.8%. The efficacy of these 8 proteins in accurate K-typing has been confirmed by a rigorous testing and the method has been automated as K-PAM (www.iith.ac.in/K-PAM/). Testing also indicates that the use of multiple genes/proteins helps in reducing the K-type multiplicity, distinguishing the K-types that have identical K-locus (like KN3 and K35) and identifying the ancestral serotypes of Klebsiella spp. K-PAM has the facilities to O-type using Wzm (ARS = 85.7%) and Wzt (ARS = 85.7%) and identifies the hypervirulent Klebsiella species by the use of rmpA, rmpA2, iucA, iroB and peg-344 marker genes. Yet another highlight of the server is the repository of the modeled 11 O- and 79 K- antigen 3D structures.A computational method has been developed to distinguish the  Klebsiella species (spp) cause a wide-range of transmissible infections 2. Two cell surface associated glycoconjugates, namely, the capsular polysaccharides (CPS) and lipopolysaccharides (LPS) are the major virulence factors of Klebsiella spp 4. While the O-antigens form the outermost component of LPS, the CPS or K-antigens form the bacterial capsule. Klebsiella strains can effectively be discriminated based on the K-  and O- serotyping 3. Currently, 141 K-types and 13 O-types are used in the serotyping of Klebsiella spp.Klebsiella spp utilize Wzx/Wzy dependent pathway for the CPS biosynthesis and surface expression, which begins with the synthesis of the nucleotide sugar precursors corresponding to a particular K-type and the assembly of the repeating unit in the cytoplasmic region with the help of the sugar specific glycosyl transferases (GTs)  is galactose) or by WcaJ  7. Followed by this, the flipping of the repeating unit to the periplasmic side takes place with the help of the flippase, Wzx. Subsequently, the repeating unit polymerization is facilitated by the Wzy copolymerase 9. Finally, Wza (an outermembrane translocon), Wzc (a tyrosine autokinase) and Wzb (a phosphatase) synergistically transport the CPS onto the bacterial surface. The surface expressed CPS is then anchored onto the Klebsiella outer membrane protein Wzi  varies across the Klebsiella spp and is used in the serotyping. The O-antigen synthesis takes place in the cytosol in a similar way as the CPS biosynthesis through the participation of several O-antigen specific GTs. After polymerization, the O-antigens are transported to the periplasmic-leaflet of the inner membrane by an ABC transporter that comprises two transmembrane domains (TMDs) (termed as Wzm) and two nucleotide-binding domains (NBDs) (termed as Wzt) 13. This O-antigen ABC transporter system is common to most of the Gram-negative bacteria. Intriguingly, the ABC transporter corresponding to some of the O-types  has an additional carbohydrate-binding domain (CBD) that is fused to the C-terminus of the NBD 16.wzi or wzc genes of the CPS biosynthesis gene cluster 17 and O-typing using wzm or wzt genes involved in the O-antigen transport are quite common 19. K- & O- typing using the arrangement of genes in the corresponding biosynthesis locus is also shown to be successful 20. Although the potential use of the other cps locus genes (other than wzi & wzc) in K-typing has been indicated previously 21, there is no detailed investigation on their genotype-serotype specificity and its utilization in the serotype prediction of Klebsiella spp. However, the genes present in the cps locus vary between different K-types 22 due to the variation in the sugar composition, steroisomoeric form, substituent and linkage. Luckily, wbaP, wcaJ, wzx, wzy, wzc, wzb, wza and wzi are common among all the K-types 22 as these genes are essential for the assembly, transport and surface anchorage of the K-antigen. One can envisage that among these eight genes, wbaP, wcaJ, wzx, wzy and wzc may be highly K-type specific due to their participation in the assembly of the repeating unit and its multimerization. Further, wbaP and wcaJ are mutually exclusive. Thus, K-typing with the use of these eight genes may be a reliable and time saving option, instead of considering all the cps locus genes (there are 508 unique protein-encoding genes) 22 for K-typing. In this context, we have classified the gene and protein sequences of wzi, wza, wzb, wzc, wzx, wzy, wbaP and wcaJ according to different Klebsiella spp K-types and demonstrated their importance in K-typing. A detailed analysis clearly indicates that the Wzx, Wzy and WbaP sequences are highly reliable for K-typing compared with other protein sequences. Owing to the fact that WbaP and WcaJ are mutually exclusive, Wzy and Wzx are found to be proficient K-type specific markers compared with other protein sequences mentioned above. Further, the use of multiple gene/protein sequences in reliable K-typing has been demonstrated. Similarly, O-typing of Klebsiella strains using wzm or wzt genes has been successfully tested.Capsule typing (or K-typing) using Klebsiella species K-/O-typing web server, namely, K-PAM (Klebsiella species serotype predictor and surface antigens modeler) (www.iith.ac.in/K-PAM/). The server accepts both the amino acid and nucleotide sequences corresponding to a single gene or multiple genes or the entire cps locus or the whole genome. The server also has the 3D structural repository of 75 K  21 and 11 O  23 antigens. The modeled K- and O- antigen repository may be useful in the perspective of designing polyvalent anti-Klebsiella vaccines 27 against a greater number of Klebsiella spp.Aforementioned approach has been implemented as an automated Klebsiella pneumoniae (hvKp) pathotype from the classical Klebsiella pneumoniae (cKp) with the use of rmpA, rmpA2, iucA, iroB and peg-344 genes (these genes are absent in the cKp) 29. Unlike cKp, hvKp causes tissue-invasive infections in healthy individuals and also spreads through community transmission 29. High mucoviscocity, higher production of CPS, aerobactin and siderophores are the characteristics of hvKp 30.In addition to the above, the server has the feature to distinguish the hypervirulent Klebsiella spp.Thus, the K-typing methodology developed and implemented as an automated web server in the current investigation would act as a rapid and accurate in silico diagnostic tool for the seroepidemiological and clinical investigations through aiding in the identification and monitoring of the Klebsiella spp in K-type prediction, wzi, wza, wzb, wzc, wzx, wzy, wbaP and wcaJ gene and their protein sequences (https://iith.ac.in/K-PAM/doc.php) are collected from the NCBI or from the Kaptive web 20 and classified according to 141 K-types. These K-types include 77 K-antigens  of Klebsiella spp whose capsule types are defined serologically 21  arrangement. The latter is known as the KL series  22. It is noteworthy that the K1–K81 K-types are also synonymously referred to as KL1–KL81 locus types. However, the sugar compositions of the remaining antigens in the KL series are unknown. Similarly, the wzm and wzt genes that are involved in the transport of Klebsiella O-antigens are being employed in the O-typing 19. Thus, wzm and wzt gene and the corresponding protein sequences are collected and classified according to their O-type (https://iith.ac.in/K-PAM/doc.php).To effectively use the eight K-locus genes of 31 to derive the information about their sequence diversity among different K-/O-types. Subsequently, the region specific diversity of the ten proteins has been analyzed individually from the amino acid sequence logo generated using Weblogo3 32.The multiple sequence alignment (MSA) and the percentage identity matrix (PIM) between the protein sequences corresponding to different K-/O-types have been generated individually for all the 10 proteins using ClustalOmega Although PIM provides the information about the degree of diversity for the eight different CPS proteins across different K-types, it may not reflect the specificity towards different K-types as it simply contains the sequence identity information. Thus, to quantify the ability of the eight K-antigen  and two O-antigen (Wzm and Wzt) biosynthesis/transport proteins in accurate serotyping , a reliability score (RS) has been introduced. The RS for each protein is estimated individually for 141 K-serotypes and 13 O-serotypes based on their ability in predicting a unique serotype when searched against the reference dataset using a 98% sequence identity cutoff:For the RS calculation, only the unique datasets  from each K-type is retained in the reference database. Note that the cutoff is chosen to be 98% as most of the non-identical sequences corresponding to the same serotype falls above 98% sequence identity and indeed, reducing the cutoff may reflect uniformly in the RS score of all the proteins. Thus, it may not affect the reliability trend. The “expected number of prediction”  has been constructed by fetching the FASTA format sequences from the NCBI  and are grouped according to 141 distinct K-serotypes. The database consists of 1345 and 1095 non-redundant gene and protein sequences respectively corresponding to 8 genes in the cps locus. To perform O-type prediction, 42 wzm and 44 wzt gene sequences and 21 Wzm and 23 Wzt protein sequences that are classified according to thirteen O-types have been incorporated in the database.A local database comprising https://www.iith.ac.in/K-PAM/) that hosts K-/O-types prediction contains the database of the reference sequences that are classified according to different K-/O-types.A Linux based web server, namely, K-PAM  or proteins can be used as the input for K-type prediction. Additionally, the user has the option of directly specifying the GenBank ID. As the whole genome sequencing (WGS) is becoming more popular nowadays, the server also accepts WGS sequence in FASTA or FASTQ formats from a single or multiple (contigs) files.The gene or protein sequences (FASTA format) corresponding to one or more CPS genes , which indicates that, in 85% of the cases, the sequences from a same K-type have the sequence identity above 98%. However, about 15% of the sequences have the identity below 98% and above 90% within the same serotype. Thus, to improve the prediction accuracy, when no serotype is found for 98% identity cutoff, the server further reduces the cutoff sequence identity to 90% in a stepwise manner (reducing the sequence identity by 2% in each round) to fetch the appropriate serotype. If no hit is observed using the above cutoff criteria, the server doesn’t report any K-type. It is noteworthy that in a similar fashion, K-typing using the gene sequences has been performed by considering 95% and 90% as the highest and the lowest sequence identity cutoffs respectively.When a single protein is given as the input, the server directly runs the BLAST To make the K-type prediction more robust, an option is also given to use the multiple genes/proteins for the prediction. In this case, the server automatically does the BLAST search and identifies the genes/proteins present in the query. As described above, the K-typing is performed for the individual proteins and is reported along with a relative reliability score . Such a combined K-typing strategy may be more promising in terms of reducing the false positive K-typing , especially in the case of less divergent Wzi and Wza sequences. When multiple K-types are predicted from different proteins, the common K-type predicted from the individual protein sequences is designated as the K-type (marked as (1) in Fig. wzm and wzt) and protein (Wzm and Wzt) sequences. Sequence identity cutoff criteria used for K-typing are followed in O-typing also. As the Wzm and Wzt sequences of O1, O2 and O2ac are highly similar (> 98%), WbbY and WbbZ sequences corresponding to O1 and O2ac have been used to distinguish them from each other as well as from O2 as suggested in the earlier studies 35. Similarly, WbdA and WbdD sequences have been added to the reference database to identify the sub-types of O3 serotype (O3/O3a & O3b) 36.Similar to K-typing, O-typing can be done using both gene (cps locus protein sequences and 45 Wzm & Wzt sequences). Similarly, to give emphasis on a protein that gives more reliable K-type compared to the other protein(s) during the multiple protein based K-typing, a relative reliability factor (RRF)  has been introduced by dividing 95%/98% = 0.97 (see the section “K-typing using multiple genes/proteins”). Thus, a K-type identified above 95% is given less weightage compared with a K-type, which is identified above 98% by simply multiplying the RS (estimated for each protein in an every new prediction) Eq.  of the fF) Table  has beenThus, the K-type prediction from the protein that has the highest ARS Table . Thus, bThe serotype prediction accuracy is validated initially by considering the sequences with known serotypes as test cases. For this, the sequences in the databases are divided into a reference dataset (consists of 141 sequences) and a test dataset (consists of 70 sequences) to corroborate the proposed algorithm. The testing against the reference dataset is done using the test dataset as a query. Further, testing is done for the sequences whose serotype is undefined in the NCBI database.Klebsiella spp form the classical Klebsiella spp using the marker genes iroB, iucA, rmpA, rmpA2 and peg-344 which are reported as hypervirulent Klebsiella strain markers in the earlier investigations 29. As per a recent definition, a strain is identified to be hypervirulent, if it possesses any 4 of the above genes 37. Based on these definitions, the server diagnoses a strain to be hvKp if 4 out of the above 5 genes are present. Additionally, if one or more of the above genes are present, the server reports that the strain has the potential to be a hvKp pathotype. It is worth noting that iroB, iucA, rmpA & rmpA2 and peg-344 genes correspond to the loci of salmochelin siderophore, aerobactin siderophore, hypermucoidy and putative transporter respectively 38. The reference dataset of K-PAM has these gene sequences and a sequence identity cutoff criterion of 60% is used to identify the presence of these genes in the query sequence.K-PAM also has the feature to distinguish the hypervirulent https://www.iith.ac.in/K-PAM/kpam_doc/sk_pam.php). This standalone version has the K-typing, O-typing and hypervirulent strain identification features. The K-PAM API generates the serotype prediction summary in the CSV file format.A standalone version (application program interface (API)) of K-PAM is also available for analyzing the large datasets. It has the option to upload multiple files. A compressed file corresponding to Mac and Linux operating system can be downloaded from the K-PAM web server  molecular modeling software 45 using the parameters derived from CGenFF  46. CGenFF encompasses the parameters for a wide range of drug-like molecules, chemical groups present in the biomolecules and heterocyclic scaffolds 46. CHARMM36 carbohydrate forcefield is used in the minimization of unmodified sugars. Minimization step includes 1500 steps of steepest descent followed by 1500 steps of adopted basis Newton–Raphson energy minimization method with a non-bonded cut-off of 16 Å. During the minimization, Generalized Born with a simple switching has been used to incorporate the solvation effect implicitly 48.In addition to the serotype prediction, the server also has the database of the modeled 3D structures of 75 K- and 11 O-antigens, the sugar composition and the glycosydic linkages within/between the repeating units, which are collected from the previous experimental studies Klebsiella K-antigen 3D structures are modeled and a local database has been created and grouped according to Klebsiella species (https://iith.ac.in/K-PAM/k_antigen.html) 49. Modeled K-antigens have been given a suffix ‘KK’ (Klebsiella K-antigen) and each sugar has been given a nomenclature depending on the substitutions and modifications as described elsewhere 50. It is noteworthy that the 3D structures of K29, K42, K52, K65, K75, K76, K77 and K78 antigens are not modeled due to the unavailability of their sugar composition and linkage information.A total of 75 Klebsiella species 23. The LPS structures have been modeled by simply specifying the sugar composition and linkage using CHARMM-GUI LPS modeler 51. The modeled LPS structures consists of three parts, namely, (1) a hydrophobic lipid A (2) core oligosaccharide linked to lipid A and (3) an O-antigen (attached to the core oligosaccharide), a common feature of the Enterobactericeae family. The core polysaccharide is usually divided into inner and outer cores, wherein; the inner core is highly conserved in the Enterobactericeae family.In addition, the server has the repository of 11-modeled LPS structures of Both the O- and K- antigens can either be visualized in the JSmol web-browser or can be downloaded and viewed by any molecular visualization software.50: rigid multimer modeling (RMM) and flexible multimer modeling (FMM).K-antigens consist of several hundred repeating units that are linked through a variety of glycosidic linkages accounting to their higher molecular weight. This makes it essential for the construction of a tool to generate K-antigen multimer. Considering this, two different multimer modeling options are available in K-PAM to generate K-antigen multimer following the methodology described elsewhere https://www.iith.ac.in/K-PAM/pim.html). The protein sequence logo corresponding to the dataset indicates that significant variation in Wzi and Wzc are confined to a particular region . Similarly, the diversity is distributed throughout Wzm and Wzt sequences (https://www.iith.ac.in/K-PAM/pim.html).The sequence identity matrices reveal the following order of diversity among the proteins used for the K-typing: Wzy (the lowest sequence identity between any two serotypes is 3%) > Wzx (5%) > WcaJ (27%) > Wzb (40%) > Wzc (45%) > WbaP (61%) > Wza (65%) > Wzi (80%)  and the three dimensional structural information may require in explaining the K-type specificity of the motif/domain involved in the protein’s transferase activity. As the structure of Klebsiella WbaP is unknown, we couldn’t pin point the K-type specific region of WbaP. Except for five pairs of K-types including K22 & K37 and K9 & K45, Wzc is highly specific to other K-types. Compared with Wzc, Wzb is less specific as it is identical for K1 & K4 and K15 & K52 in addition to K22 & K37 and K9 & K45. WcaJ that possesses the next ARS is identical for K16, K54, K58 & KL113 .A careful inspection indicates that the K-type specificity is accumulated in the middle region of Wzi  that is flanked by more conserved regions on either side  and the K-typing is done for all the three fragments individually  of Wzc  or wzc (or Wzc) are in general used in the K-typing 53, K-PAM has the option of using multiple genes/proteins for serotyping as it increases the prediction accuracy by removing the false positives. For instance, as the Wzi of K9, K38 and KL105 possess a very high sequence identity between them, when a Wzi query sequence of K38 is submitted, the server reports all the three K-types as the potential serotypes. Similarly, when the Wzc sequence of K38 alone is submitted, the server reports K38 and KN3 as predicted K-types. On the other hand, incorporation of Wza along with Wzi results in 100% prediction accuracy (https://iith.ac.in/K-PAM/doc.php). Most interestingly, K9 and K45 that have above 98% sequence identity for Wza, Wzb and Wzc can easily be distinguished on the basis of Wzx that is unique between the two (see the section “Serotyping of unclassified K- and O-types”). Additionally, as WbaP and WcaJ are mutually exclusive genes, K9 and K45 can be discriminated based on the presence of WbaP and WcaJ respectively. Thus, using the multiple proteins increase the efficiency of K-typing . Finally, a graph illustrating the reliability of prediction is displayed alongside the predicted K-type, wherein, the RS  and K35 (Genbank ID: AB924573.1) are 87%, 91%, 87%, 91%, 85%, 86% and 88% respectively. Subsequently, a more rigorous testing has been carried out by considering 162 dataset from the published literature 20. The results clearly show that K-PAM accurately predicts the serotype  K-types (KL101 to KL165) are found to have a sequence identity with the K1-K81. For instance, Wza, Wzb, Wzc, Wzy and WcaJ of KL104 shares 99%, 100%, 99%, 97% and 99% sequence identity respectively with K30. However, the Wzi sequence has only 96% sequence identity and Wzx sequence does not share any identity with K30. Similarly, KL106, KL135, KL142, KL148 and KL163 share close sequence identity with K22, K40, K44, K36 and K21 respectively Table .rfb) (see Methods).Both Wzm (the lowest sequence identity between any two serotypes is 19%) and Wzt (the lowest sequence identity between any two serotypes is 33%) are highly divergent across the 13 O-types, thus, have become the highly suitable candidates for the prediction of O-types. Notably, the ATP binding domain of Wzt is absent in O1, O2, O8 and O9 types. Intriguingly, the ARS corresponding to Wzm (85%) and Wzt (85%) indicates that these proteins may exhibit a less O-type prediction accuracy  taken from NCBI (Table https://iith.ac.in/K-PAM/doc.php for single protein based predictions). The importance of using Wzi and Wzc sequences together has been demonstrated by considering the NCBI accession numbers CZQ24079.1 (Wzi), BAI43775.1 (Wzi), CZQ24082.1 (Wzc) and BAI43778.1 (Wzc) (https://iith.ac.in/K-PAM/doc.php). For instance, the multiple K-types , which are predicted from Wzi (CZQ24079.1) alone has been narrowed down to K15 with the inclusion of Wzc (CZQ24082.1). Similarly, the K-types predicted individually for BAI43775.1 (Wzi) are K9 & KL105 and BAI43778.1(Wzc) are K9 and K45. Thus, K9 is predicted to be the K-type as it occurs in both the cases. Interestingly, K-PAM predicts K11 as the serotype for Genbank accession ID LT603705 with 100% reliability from multiple genes that was wrongly annotated as KL129 earlier 54  information derived using PCR technique  is accumulated in the NCBI database 55. Thus, K-PAM will be useful in improving the serotype epidemiology and pathophysiological insights about the Klebsiella spp.Although the server is capable of handling the whole genome sequencing that is becoming very popular and efficient, it can be useful in the serotyping of several unclassified wbbY and wbbZ genes that are located outside the O-antigen biosynthesis locus are additionally used to distinguish O1, O2 and O2ac serotypes 35. Similarly, wbdA and wbdD genes are used to distinguish O3 variants.O-typing using Wzm and Wzt has also been predicted and validated as described above by considering the NCBI accession ID CZQ25314.1 as a case in point Figure . In facthttps://iith.ac.in/K-PAM/doc.php). Another example in this category is Genbank ID: CP035214.1.K-typing methodology implemented in K-PAM is capable of not only identifying the ancestral K-type(s) that has emerged through cross-reaction, but also, a totally new K-type. For instance, the sequence identity of Wzc, Wzy, and WbaP proteins fall below 95% for Genbank ID CP029128.1, thus, the K-type predicted from them is not considered. Nonetheless, Wzi has 99.3% sequence identity with K74 and Wza has the sequence identity of 95.8% with both KL127 and KL131. Thus, K-PAM reports CP029128.1 as a new serotype (When a particular K-type is reported dominantly from multiple (at least 3) protein sequences except one or two, K-PAM reports the new serotype as a variant of the dominant K-type. For the Genbank ID CP020358.1, the nearest K-type predicted from all the 7 proteins  is K29, but, the sequence identity between the reference and query sequences of Wzi is very poor. In this case, K-PAM predicts the serotype as the variant of K29. Interestingly, for the Genbank ID NZ_AP014950.1, Wzx, Wzy, Wzc, Wzb and WcaJ predict the serotype as K67. However, Wzx that has a higher ARS compared with the other proteins has only 93% sequence identity between the query and reference sequences. Thus, serotype is predicted here as the variant of K67. In addition, Wzi and Wza in the query sequence have less sequence identity (92% and 93% respectively) with K67 reference protein sequences. Thus, the methodology implemented here will also be useful in identifying the newly emerging K- or O-types.56 and Kaptive web 20 are available for Klebsiella spp, they are either based on the arrangement of K-antigen gene cluster or based on a single gene sequence. Further, as K-PAM predicts the K-type based on multiple gene/protein sequences as well as from fragment based method, it can easily identify the antigen variants and their ancestor(s). Importantly, K-PAM precisely distinguishes the K-types that have identical cps locus arrangement .Although in silico K-typing tools such as BIGSdb Klebsiella strains has been demonstrated by considering several clinically important strains 61. Figure Klebsiella variicola strain as an example 60. Results indicate that the strain has all the hypervirulent marker genes . Table The efficacy of K-PAM in identifying the hypervirulent https://iith.ac.in/K-PAM/k_antigen.html), the database redirects the user to a webpage that comprises the chemical and schematic representations of the Klebsiella spp CPS repeating unit. The page also contains an interactive Jsmol applet for the visualization of 3D structure of the K-antigen along with the provision to download the coordinates in protein databank format. The polymeric form of the K-antigens can be generated by choosing either rigid multimer modeling .Although 2 major K-antigen classifications can be derived based on the presence of initializing galactose and glucose transferases WbaP and WcaJ respectively 62) that has been deposited in the repository can be accessed through O-antigen menu bar. The LPS structures can either be visualized in the Jsmol applet or be downloaded.LPS models that correspond to O-antigens, namely, O1, O2, O2aeh, O3, O4, O5, O7, O8 and O12  in K-typing. The result indicates that WbaP/WcaJ, Wzx, Wzy and Wzc can be effectively used for K-typing due to their higher K-type specificity compared with Wzb, Wza and Wzi. The use of multiple genes/proteins individually for the K-typing reduces the K-type multiplicity. Although cps locus organization has become popular in K-typing due to the distinct cps locus arrangement between the different K-types 22, the missing information about one or more genes may mislead the K-typing. Further, the cps locus arrangement based K-typing may have a limitation when two K-types have an identical locus arrangement, but, have two different K-antigen repeating units, as seen in KN3 and K35. Thus, eight CPS gene/protein based K-typing approach developed, implemented (as K-PAM web server) and tested here would facilitate the accurate K-typing. The robustness of the multiple genes/proteins based K-typing approach in reducing the false-positives, reporting a variant K-type and identifying the emergence of a new K-type has been illustrated by considering several clinically important sequences. K-PAM can extensively be used to extract the K-type of the untyped Klebsiella strains deposited in NCBI to aid in the betterment of seroepidemiological knowledge. To our knowledge, this is the first study that has precisely investigated the serotype-genotype relationship using only 8 cps locus genes  in any bacterial species that use Wzx/Wzy-dependent pathway. Thus, this method can be extended to other bacterial species for rapid and accurate K-typing. Similarly, Wzm and Wzt facilitate the prediction of O-type with 100% accuracy. The server also hosts an additional feature that distinguishes the hypervirulent Klebsiella strain from the classical strain. Further, the server has the capability to accept the whole genome sequences in a single file or multiple files (contigs). The standalone version of K-PAM has an additional feature of handling the multiple query sequences (as multiple input files) in one go. Thus, K-PAM would be a useful diagnostic tool in the seroepidemiological and pathophysiological investigations of Klebsiella spp infections. The 3D repository of the modeled K-/O- antigen structures which are accessible through online may be useful in the modeling studies that require a good starting model of Klebsiella K-/O-antigen(s) to facilitate the design of anti-Klebsiella vaccines.A detailed analysis has been carried out to test the efficacy of eight Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.


S473 at the 2- and 4-cell stages and of p-ERK1/2T202/Y204 nuclear translocation at the 2-, 4-, and 8-cell stages. L-Pro improvement in embryo development involves mechanisms analogous to those seen with Pro-mediated differentiation of mouse ES cells, which is also stereoselective, dependent on transporter uptake, and activates Akt, ERK, and mTORC1 signaling pathways.Groups of amino acids, and some selected amino acids, added to media used for culture of pre-implantation embryos have previously been shown to improve development in various ways including survival to the blastocyst stage, increased blastocyst cell number and improved hatching. In this study, we cultured 1-cell mouse embryos for 5 days to the hatching blastocyst stage in isosmotic medium (270 mOsm/kg) at high density (10 embryos/10 μL), where autocrine/paracrine support of development occurs, and low density (1 embryo/100 μL), where autocrine/paracrine support is minimized and development is compromised. When 400 μM L-Pro or 1 mM L-Gln was added to embryos at low density, the percentage of embryos reaching the blastocyst stage and the percentage hatching increased compared to low-density culture without these amino acids, and were now similar to those for embryos cultured at high density without amino acids. When L-Pro or L-Gln was added to embryos at high density, the percentage of embryos reaching the blastocyst stage didn’t change but hatching improved. Neither embryo culture density nor the presence of these amino acids had any effect on blastocyst cell number. D-Pro and the osmolytes Gly and Betaine did not improve embryo development in low- or high-density culture indicating the mechanism was stereospecific and not osmotic, respectively. L-Pro- and L-Gln-mediated improvement in development is observed from the 5-cell stage and persists to the blastocyst stage. Molar excess of Gly, Betaine or L-Leu over L-Pro eliminated improvement in development and hatching consistent with them acting as competitive inhibitors of transporter-mediated uptake across the plasma membrane. The L-Pro effect is dependent on mTORC1 signaling (rapamycin sensitive) while that for L-Gln is not. The addition of L-Pro leads to significant nuclear translocation of p-Akt In partmpaction , allows mpaction .2+ transients   and this effect is mitigated in low-density (LD) culture  . A numbeansients , which achannels , 2009. Bchannels  thereby S473 and p-ERK1/2T202/Y204 and the improvement in development is dependent on mTORC1 signaling. These results are analogous to L-Pro’s growth-factor-like stimulation of differentiation of mouse ES cells, which is also dependent on transporter-mediated uptake and involves activation of the Akt, ERK and mTORC1 signaling pathways  mice  were used in accordance with the Australian Code of Practice for Use of Animals in Research and the study was approved by the University of Sydney Animal Care and Ethics Committee . Mice were housed under a 12-h light:12-h dark cycle. Female mice (4-10 weeks old) were induced to ovulate by intraperitoneal injection of 10 IU pregnant mare serum gonadotrophin  followed 48 h later by 10 IU human chorionic gonadotrophin (hCG) and paired with males overnight. Female mice were euthanized by cervical dislocation 20-22 h after hCG injection. Zygotes were isolated as described previously . The med2) to day 6 of development, either in dishes (for HD culture) or round bottom, 96-well plates (for HD and LD culture) . HD culture was performed by placing 10 embryos into a 10 μL drop of medium under mineral oil (Sigma). LD culture was performed by placing individual embryos into 100 μL medium in each well of a 96-well plate. Medium was pre-equilibrated at 37°C with 5% CO2 for a minimum of 2 h before adding the embryos. Embryos were scored for developmental stage every 24 h. Hatching from the zona pellucida was scored on day 6 of development (144 h post-hCG). The medium was not changed over the culture period unless indicated.Embryo culture was performed in mHTF containing 0.3 mg/mL BSA and with the different NaCl concentrations and osmolalities described above. L-Gln was omitted from all media unless stated. Embryos were cultured from the zygote stage (day 1 of development) for 120 h  in PBS/polyvinyl alcohol  for 30 min, followed by permeabilization in PBS/PVA + 0.3% Triton X-100 for 30 min at RT. Blastocysts were then mounted on coverslips in Vectashield + DAPI (Vector Labs) and imaged by confocal microscopy (Zeiss LSM510 Meta). The number of nuclei stained with DAPI was counted to give the total cell number.After being cultured in various treatments to the 2-, 4- or 8-cell stages, embryos were fixed in 4% PFA in PBS/PVA for 30 min at room temperature and then washed twice in PBS/PVA.In order to eliminate variability between samples during phosphoprotein staining, embryos in different treatment groups underwent the staining process together. To achieve this, the membranes of embryos cultured in control medium were first labeled with concanavalin A Alexa Fluor 647  (1:50 dilution in PBS/PVA) for 30 min in the dark at room temperature. Then, embryos in control and treatment groups underwent the rest of the staining process together in the same aliquot of phospho-antibody and were identified later by the presence or absence of ConA staining.T202/Y204 , 1:50 for p-AktS473 , 1:200 for goat anti-rabbit IgG Alexa Fluor 488 . For primary antibodies, embryos were incubated overnight at 4°C and then washed three times in PBS/PVA + 0.7% BSA + 0.1% Tween-20. Embryos were then incubated in secondary antibody in the dark at room temperature for 2 h and again washed three times in PBS + PVA + 0.7% BSA + 0.1% Tween-20. Embryos were transferred to a drop of Vectashield + DAPI and DAPI-stained nuclei and secondary antibody fluorescence were visualized on a confocal microscope with a 40× objective using 405 and 488 nm Argon lasers, respectively. ConA fluorescence was visualized using a helium-neon laser at 633 nm. Embryos were scanned through the plane of the nuclei for the purpose of measuring the relative fluorescence intensity in the nuclei compared to the cytoplasm using ImageJ software.Embryos were permeabilized in PBS/PVA + 0.3% Triton X-100 for 30 min at room temperature, then washed in PBS/PVA before being blocked in PBS/PVA + 0.7% BSA + 0.1% Tween-20 for 30 min at room temperature. Primary and secondary antibodies were diluted in PBS + PVA + 0.7% BSA + 0.1% Tween-20, as follows: 1:200 for p-ERKFor each blastomere, average nuclear fluorescence intensity was determined from independent measurements in three square areas of the same size excluding the nucleoli. The same measurements were performed in the cytoplasm. The ratio of the average nuclear intensity to the average cytoplasm intensity was calculated to indicate the extent of nuclear phosphoprotein localization. Mean intensity measurements were performed blind by randomization of files.t-test.Culture experiments were performed at least three times, with at least 15 embryos in each treatment group, and the data pooled. Proportions were compared using Fisher’s exact test or Chi-squared analysis, with Bonferonni corrected significance levels for multiple comparisons. Cell counts from at least three independent experiments were pooled and expressed as mean ± SEM and data were compared using ANOVA, as described in the figure legends, using Graphpad Prism v7. Fluorescent nuclear/cytoplasmic ratios were pooled from at least three independent experiments and data were compared using an unpaired Media with an osmolality of 270 mOsm/kg was used in this study as it resulted in optimal blastocyst development and blastocyst hatching in both HD (10 embryos/10 μL) and LD (1 embryo/100 μL) culture conditions . Mouse zFor LD at higher osmolality (300 and 330 mOsm/kg), development to the blastocyst stage and hatching were reduced. HD culture was able to partially overcome these effects . SimilarFor embryos cultured at LD in 270 mOsm/kg medium, the addition of either 400 μM L-Pro or 1 mM L-Gln increased the percentage of blastocysts formed and the percentage of blastocysts that hatched compared to embryos grown in the absence of amino acids . In factA range of other amino acids – the D-isomer of Proline (D-Pro), L-Ala, and the osmolytes Gly and Betaine – failed to increase blastocyst development in LD culture at 270 mOsm/kg  and all In HD culture at 270 mOsm/kg, none of the amino acids improved blastocyst development or increased cell numbers compared to embryos cultured without amino acids . HoweverAs noted previously, in LD culture under isosmotic conditions (270 mOsm/kg), only added L-Pro or L-Gln increased the percentage of blastocysts that formed , and no Zygotes cultured at LD in isosmotic medium were scored for stages of development each day over the 5-day culture period . Almost The timing of the beneficial effect of L-Pro on development was investigated by culturing embryos in the presence of L-Pro over restricted time windows. Embryos cultured in the presence of L-Pro only for the first 24 h (up until the late 2-cell stage) developed no better than embryos cultured in the absence of amino acids . In contWe have previously shown that L-Pro induces the differentiation of mouse embryonic stem cells (ESCs) to primitive ectoderm and that this differentiation can be inhibited by the addition of molar excess of selected amino acids, which competitively inhibit plasma-membrane transport via the amino-acid transporter SNAT2 . SimilarWhen the highly selective mTORC1 inhibitor rapamycin (10 nM) was added to embryos in culture from the 2-cell stage, the L-Pro-mediated improvement in development to the blastocyst stage was prevented . At thisS473 at the 2- and 4-cell stages but not at the 8-cell stage, while nuclear translocation of p-ERKT202/Y204 was increased at all 3 of these stages of development. L-Gln added to zygotes also stimulated nuclear translocation of p-AktS473 at the 2-cell stage but had no effect on nuclear translocation of p-ERKT202/Y204. Further investigation is needed to confirm whether L-Gln can act in a growth factor like manner in pre-implantation embryos.Our results show that L-Pro and L-Gln each improves the development of embryos to the blastocyst stage when embryos are cultured at LD . The effInstead, our data suggest that L-Pro or L-Gln added to embryos cultured at LD under isosmotic conditions act in a growth factor- or autocrine-like manner to improve development. Consistent with this, the improvement in the development of embryos in LD culture in the presence of either of these specific amino acids was similar to that seen at HD in the absence of amino acids, where autocrine/paracrine support is available . A numbeIn vivo, the developing embryo, as well as secreting its own amino acids, is bathed first in tubal and then uterine fluid. The concentrations of amino acids in these fluids fall in the low millimolar to submillimolar range; i.e., similar to the concentrations of L-Pro and L-Gln used here   . Thus, alestones . SufficimTOR–/– mouse embryos exhibit reduced proliferation and differentiation of the inner cell mass and trophectoderm, and embryonic lethality just prior to implantation   . ConsistThe effect of amino acids in terms of hatching was different to that in terms of development to the blastocyst stage in two important respects: (i) L-Pro or L-Gln improved hatching not just in LD but also in HD culture . (ii) L-in vitro .MM and MD were responsible for writing and preparing the drafts and final manuscript. MD designed and prepared the figures, with legends, based on the data generated by SO, MatZ, MarZ, and RZ. MM, MD, and MT conceived the project and designed the original experiments. SO was the principal contributor of data for this research. All authors read, edited and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Community-acquired pneumonia (CAP), especially pneumococcal CAP (P-CAP), is associated with a heavy burden of illness as evidenced by high rates of intensive care unit (ICU) admission, mortality, and costs. Although well-defined acutely, determinants influencing long-term burden are less known. This study assessed determinants of 28-day and 1-year mortality and costs among P-CAP patients admitted in ICUs.Data regarding all hospital and ICU stays in France in 2014 were extracted from the French healthcare administrative database. All patients admitted in the ICU with a pneumonia diagnosis were included, except those hospitalized for pneumonia within the previous 3 months. The pneumococcal etiology and comorbidities were captured. All hospital stays were included in the cost analysis. Comorbidities and other factors effect on the 28-day and 1-year mortality were assessed using a Cox regression model. Factors associated with increased costs were identified using log-linear regression models.p = 0.0002), liver diseases , and the illness severity at ICU admission. Compared with non-ICU-admitted patients, ICU survivors remained at higher risk of 1-year mortality. Within the following year, 38.2% (516/1350) of the 28-day survivors required at least another hospital stay, mostly for respiratory diseases. The mean cost of the initial stay was €19,008 for all patients and €11,637 for subsequent hospital stays within 1 year. One-year costs were influenced by age , chronic cardiac , and respiratory diseases .Among 182,858 patients hospitalized for CAP in France for 1 year, 10,587 (5.8%) had a P-CAP, among whom 1665 (15.7%) required ICU admission. The in-hospital mortality reached 22.8% at day 28 and 32.3% at 1 year. The mortality risk increased with age > 54 years, malignancies (hazard ratio (HR) 1.54, 95% CI [1.23–1.94], P-CAP in ICU-admitted patients was associated with a heavy burden of mortality and costs at one year. Older age was associated with both early and 1-year increased mortality. Malignant and chronic liver diseases were associated with increased mortality, whereas chronic cardiac failure and chronic respiratory disease with increased costs.N/A (study on existing database) Community-acquired pneumonia (CAP) causes a heavy burden of illness with high morbidity, mortality, and health-related costs –5. In EuAmong hospitalized CAP, the proportion of those requiring intensive care unit (ICU) admission ranges from 5 to 40% ; it was In addition, the burden of in-hospital management of CAP, due to pneumococci (P-CAP) and other etiologies, as well as management of associated comorbidities, generates high costs. Although the short-term mortality and in-hospital costs of P-CAP in France have already been estimated , little The objectives of this study were to ascertain the overall burden associated with ICU-admitted P-CAP in France in terms of in-hospital mortality and the associated direct costs, during both the initial hospital stay and within the year following the P-CAP onset.The study objective aimed to evaluate the burden of severe P-CAP in France related to the initial hospital stay and subsequent hospital stays within the year following the pneumonia onset, regarding in-hospital mortality, and hospital-related costs. The study is based on an exhaustive administrative registry that collects data of all hospital stays in France.Programme de Médicalisation des Systèmes d'Information (PMSI) database, the health administrative database which records all discharges from public and private hospitals in France. This registry allows the chaining of all hospital stays of individual patients.Study data were obtained from the Streptococcus pneumoniae” or B953 “Streptococcus pneumoniae as the cause of diseases classified elsewhere” and pneumonia. Patients with a hospital stay with a diagnosis of P-CAP within the previous 3 months were excluded. The severity of acute illness at ICU admission was reported through the Simplified Acute Physiology Score II (SAPS II), mandatory for all ICU stays. Additional file All patients admitted to ICUs with a diagnosis of P-CAP were included. The International Statistical Classification of Diseases, 10th Revision (ICD-10), used for CAP diagnosis and comorbidities are detailed in the Online Data Supplement. A CAP-related hospitalization was any hospitalization with a principal diagnosis of pneumonia or a secondary diagnosis of pneumonia if the principal diagnosis was respiratory failure or sepsis. For P-CAP, the diagnoses were based on ICD-10 code J13: “Pneumonia due to All hospital stays, encompassing those related to the initial ICU admission as well as subsequent hospitalizations within the following year (detailed in the supplement), were included in the cost analysis, with the details of inclusion stay costs (including the supplement related to ICU) and 1-year follow-up costs. Healthcare consumption items taken into account are described in Additional file Costings were restricted to direct in-hospital costs and determined from the perspective of the French social security system, according to the methodological rules of the French Technical Agency for Information on Hospitalization. Valuation of hospital stays included the 2014–2015 tariffs of the GHS (using upper and lower limits), with supplements and additional expenses related to drugs and medical devices. For private hospitals, clinicians' fees were added. Costs were expressed in 2016 Euros.All hospital stays were considered to be independent and included into the analyses. Standard descriptive statistics were performed for the whole cohort. Data were summarized as frequencies and percentages for categorical variables and medians with interquartile ranges or means with standard deviations for continuous variables.p value < 0.1) were included in the multivariate analysis with backward selection.Log-linear regression models were constructed to explore predictive factors associated with costs in adults. Factors significantly associated with the outcomes assessed in the univariate analysis  and late mortality (within the first year excluding the first 28 days) to respect proportional hazard hypotheses. Proportionality assumption of Cox models was checked graphically, and Schoenfeld residuals were calculated if clinically relevant.p values < 0.05 were considered significant. Data were analyzed using R .The Of a total of 182,858 patients hospitalized for CAP in France during the year 2014 and without any hospitalization for pneumonia within the previous 3 months, 10,587 (5.8%) patients had P-CAP, from which a small minority of patients required ICU admission  , chronic cardiac failure , malignant disease   and diabetes . Alcohol and/or tobacco abuse was reported in 740 (44.4%) patients. At ICU admission, the mean (SD) SAPS II was 48  vs. 64 [53–75] years)..Overall, 516 (31.0%) patients were readmitted at hospital within the following year, most of them once . However, 25 (1.5%) were readmitted 4 times, and 25 (1.5%) patients were admitted 5 times or more. Most of the readmissions were due to diseases of the respiratory system  and circulatory system (13.6%). The mean (SD) cumulated duration of the stays during the following year was 10.7 (14.3) days. Although the mean number of stays was rather similar regardless of their age-group, the cumulative duration increased with age, from 9.0 (10.9) days for the 55-to-64 age-group to 11.3 (13.7) days for the 65-to-75 age-group  , compared with 13.4% (1196/8922) of those hospitalized not admitted in ICU, and their in-hospital mortality occurred earlier, at a median [IQR] of 0.7 [0–2] months versus 1.7 [0–5] months for the other inpatients. Among ICU-admitted P-CAP patients, 70.4% of deaths occurred within the first 28 days and 9.5% during the second and third months  and chronic liver diseases   cost of the initial hospital stays was €19,008   , whereas costs were significantly decreased for the most severely ill patients , in that study that contrasts to our study where diabetes had no prognostic value in multivariate analysis. The frequent complication of diabetes with chronic cardiovascular diseases may explain why diabetes itself is not eventually associated with prognosis in our multivariate model.The 28-day mortality rate of our study is consistent with that reported by others. In a multicenter French study on 614 ICU-admitted patients with severe P-CAP, the mortality rate was 18.9% . Age, geAfter the initial ICU stay, the 1-year in-hospital mortality was 12% and increased with age and comorbidities including malignant and liver diseases. Our observed mortality is similar to the overall 1-year post-ICU mortality reported by a Dutch study  but loweIn our study, the mean cost of the initial ICU stay was slightly above €19,000. In a previous study conducted in France in 2011–2014 which evaluated the direct costs associated with P-CAP, the cost for the subset of the ICU-admitted patients was €14,385, that is, lower than observed in our study . With reThe 1-year hospital costs were closely linked to rehospitalizations and were experienced by one patient out of three in our study. This rate is lower than the 40% readmission rate within 3 months observed in the USA for sepsis , and theIn our study, specific comorbidities but not age were independent prognosis factors for the pneumococcal disease burden, that supports French recommendations, which are solely comorbidities driven for adult patients . This isDespite its strengths, this study has some limitations. It relies on an administrative database that mainly has budgetary purposes. Therefore, the coding of some diseases such as the P-CAP diagnosis might be suboptimal. The reliability is not that of a clinical database monitored against source data. However, we used ICD-10 of the coding system, which showed more reliability for the pneumonia diagnosis, and we combined codes for an improved accuracy , 44. In S. pneumoniae, or pneumococcal invasive infections other than CAP, or other reasons besides CAP patients for being transferred into the ICU.Another limitation is that this database collects solely in-hospital data. Therefore, we do not have any insight regarding what happened outside of the hospital, such as the out-of-hospital mortality rate. However, a French study showed that the rate of in-hospital deaths in 2008 ranged approximately 60% of deaths for the patients aged between 40 and 89 years and that these rates were stable over 15 years . TherefoAlthough severe pneumococcal pneumonia in France was associated with a survival of 81% at discharge, the Kaplan–Meier estimates of the 1-year survival were 68%. The 1-year prognosis was mainly influenced by age and comorbidities, such as chronic liver diseases and malignant diseases. The burden of severe pneumococcal pneumonia is not only a heavy mortality but also a high rate of new hospital stays within the year following the pneumonia onset. Overall, the in-hospital costs within the year following pneumonia were higher than €22,000 and significantly increased in patients with cardiac diseases and chronic respiratory diseases. Overall, severe pneumococcal pneumonia in patients with chronic comorbidities, such as malignant diseases, chronic liver, and cardiac and respiratory diseases, is associated with a heavy 1-year burden, and it might be prevented by a better compliance to the current guidelines for pneumococcal vaccination.Additional file 1. Further details on Methods, on data regarding the hospital stays (e-Table 1 and e-Table 2), and on the 1-year survival curves of patients . In Methods, further details are provided on the codes used for the identification of the pneumonia diagnosis, of the comorbidities and procedures used, and of the pneumonia etiology. e-Table 1. Characteristics of the new hospital stays within the year following the CAP-related hospitalization, overall and according to the age group, among those with at least one new hospital stay. e-Table 2. Direct costs of the initial hospital stay and of the subsequent hospital stays within the first year, by patient with pneumococcal communityacquired pneumonia and admitted in intensive care unit, overall, according to the one-year outcome, and by age group. e-Fig. 1. Comparative survival curves of patients with pneumococcal pneumonia (P-CAP) admitted in intensive care unit (ICU), (A) from the hospitalization to 3 months after  ICU-admitted patients vs. 91.7%  non ICU-admitted patients); (B), among the 3-months survivors, from 3 to 12 months after the hospitalization  ICU-admitted patients vs. 94.5%  non-ICU-admitted patients).


The 2019 novel coronavirus (COVID-19) presents a major threat to public health and has rapidly spread worldwide since the outbreak in Wuhan, Hubei Province, China in 2019. To date, there have been few reports of the varying degrees of illness caused by the COVID-19.A case of 68-year-old female with COVID-19 pneumonia who had constant pain in the right upper quadrant of her abdomen during her hospitalization that was finally diagnosed as acute cholecystitis. Ultrasound-guided percutaneous transhepatic gallbladder drainage (PTGD) was performed, and the real-time fluorescence polymerase chain reaction (RT-PCR) COVID-19 nucleic acid assay of the bile was found to be negative. PTGD, antibacterial and anti-virus combined with interferon inhalation treatment were successful.The time course of chest CT findings is typical for COVID-19 pneumonia. PTGD is useful for acute cholecystitis in COVID-19 patients. Acute cholecystitis is likely to be caused by COVID-19 . In December 2019, an outbreak of the 2019 novel coronavirus (COVID-19) occurred in Wuhan, Hubei Province, China, and rapidly spread throughout the world . Here, wOn January 31, 2020, a 68-year-old woman presented to the hospital with a 6-day history of fever, chest stuffiness and diarrhea without chills, cough, or nasal discharge. She had a history of good physical health and no underlying diseases. She had stayed with her son who had been diagnosed with 2019 novel coronavirus (COVID-19) pneumonia after business travel to Wuhan, China.9/L , differential neutrophil count of 70.7% , and lymphocyte count of 20.3% . The alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin levels were 19.0 (9.0–52.0) U/L, 27.0 (14.0–36.0) U/L, and 19.9 (3.0–22.0) μmol/L, respectively. There were elevated blood levels for C-reactive protein .Her body temperature was elevated to 38.1 °C (100.6 °F) for 6 days before her hospitalization. The real-time fluorescence polymerase chain reaction (RT-PCR) assay of pharyngeal swabs and feces was positive for COVID-19 nucleic acid upon hospitalization. The patient’s temperature on admission was normal at 36.3 °C (97.3 °F), and there were coarse breath sounds from both lungs on auscultation. Laboratory studies showed a normal white blood cell count of 3.64 × 10In the present case, the chest CT findings were typical for COVID-19 pneumonia  in the right lower lobe, which may represent hyaline membrane formation. Follow-up CT in this case demonstrated mild disease progression, as manifested by the increasing extent and multiple patchy consolidations, especially in the peripheral zones of the lungs, which may represent alveolar injury and inflammatory exudation. After symptomatic treatment, the consolidations and GGOs were almost absorbed, leaving cord-like shadows, which represent an improvement of the disease. The time course of lung changes on chest CT images of this patient were typical, as described in previous studies –8.In our unique case, the patient developed constant pain in the right upper quadrant of the abdomen and Murphy’s sign after 10 days of hospitalization, and her body temperature was elevated to 38.3 °C. Because of the typical clinical presentation, this patient was likely to be diagnosed as acute cholecystitis or cholangitis in the clinic. Acute cholecystitis was subsequently confirmed by plain abdominal CT scan. It was difficult to identify the cause of acute cholecystitis in COVID-19 patients by abdominal CT imaging. And it could potentially cause severe gallbladder perforation, subsequently it was treated with PTGD. There has not been a study to report the RT-PCR nucleic acids for bile, while sputum or feces were with the highest positive rate of RT-PCR results. The precise mechanism of acute cholecystitis in COVID-19 patients was unknown. The RT-PCR results failed to find virus of COVID-19 in the bile. So our speculation about the potential association in the COVID-19 patient developed acute cholecystitis was a possible complication of COVID-19. However it remains to envisage acute cholecystitis as a possible complication of COVID-19, pending further studies that could prove or disprove this hypothesis.It has not been reported whether the gallbladder might be vulnerable to COVID-19. We highlight a further complication possible with COVID-19. But the notable limitations of this study should be acknowledged. Only one unique COVID-19 patient presented an acute cholecystitis, the PCR test of the bile show no evidence of COVID-19 virus invasion of the gallbladder, and lack of pathological diagnosis for gallbladder tissue, which are not in favor of the possible association between COVID-19 and acute cholecystitis, it remains however possible that the infection by COVID-19 virus triggers the cholecystitis via a yet unknown mechanism.In conclusion, we report the clinical course of a female patient with COVID-19. The time course of chest CT findings is typical for COVID-19 pneumonia. PTGD is useful for acute cholecystitis in COVID-19 patients. Acute cholecystitis is likely to be caused by COVID-19.